This is a competing renewal application. The overall thrust of the project is to develop novel fluorescent sensing and imaging technology for understanding the biology of zinc, with a view to developing novel and improved diagnostics and therapies for human diseases. Evidence of zinc's role(s) in stroke, Alzheimer's disease, epilepsy, long term memory, gastrointestinal infectious disease, and type 2 diabetes has recently emerged, but a much more detailed understanding of zinc's involvement will be necessary in all these cases. Previously, we had developed protein-based fluorescence indicators with outstanding sensitivity, selectivity, and tunable kinetics;using this basic approach we were able for the first time to measure (in a resting cell) free zinc in the cytoplasm, and by use of an expressible indicator, in the resting mitochondrion. For the next cycle we propose to develop a palette of new ratiometric, expressible zinc indicators that will provide rapid, sensitive, and reliable zinc quantitation in many applications. We also are proposing to develop an accurate measurement of the expression of our biosensors in situ by microscopy;this basic approach may be useful to quantify protein expression in general. We also are proposing to apply our new sensing technology to answer questions of central importance that so far have resisted other investigators: Is hypoxia alone or the acidosis it induces responsible for the release of toxic zinc levels in ischemia? In the hippocampal formation in the brain, how much zinc ion is released in response to electrical stimulus, and how much of the zinc is taken up by the post-synaptic dendritic spines? Answers to these questions will help elucidate zinc's role in stroke (and suggest means for treatment, which currently are marginal), and in learning and memory formation. PUBLIC HEALTH RELEVANCE: The proposed work will be vital for understanding the role(s) of zinc in intractable diseases such as stroke, Alzheimer's disease, epilepsy, type 2 diabetes, and others. This novel technology will provide the basis for new diagnostics and treatments.